THE POLLINATION OF EPIPACTIS ZINN , 1757 ( ORCHIDACEAE ) SPECIES IN CENTRAL EUROPE – THE SIGNIFICANCE OF CHEMICAL ATTRACTANTS , FLORAL MORPHOLOGY AND CONCOMITANT INSECTS

A series of studies on pollination biology of the Epipactis atrorubens (Hoffm.) Besser, E. purpurata Sm., and E. palustris (L.) Crantz populations was conducted in Poland, Lithuania and Czech Republic between 2003 and 2010. The research focused on pollinators and visitors to aforementioned orchid species as well as on the chemical analysis of orchids’ nectar which was done using the GC/MS method. It was found that: 1) the type of pollinators depends on the orchid population size and the surrounding environment, where the temperature and amount of precipitation during the vegetative season are the most vital factors; 2) pollinators and visitors to the examined orchids might differ in successive growing seasons; 3) the studied Epipactis species differ in the chemical composition of their nectar and its scent, which can influence their pollination biology; 4) the tendency to autogamy observed in E. purpurata might be due to lack of pollinators in its habitats.


INTRODUCTION
Despite an ongoing research, the collected data on Epipactis differ significantly, and their breeding system still remains unidentified.As stated by Neal and Anderson (2005), the breeding system is most likely a consequence of a relatively short existence of Orchidaceae as a species, as well as its remarkable adaptability.Additionally, the chemical composition of nectar in the species of Epipactis has not been defined and there is no certainty whether it contains compounds directly responsible for attracting insects.
The Helleborines are rhizomatous orchids with a tendency towards autogamy.Yet, some literature indicates cases of geitonogamy, as well as allogamy and predominantly allogamy (Tałałaj and Brzosko 2008).Autogamic or facultative autogamic species also arouse controversy around their pollination ecology.It is still not clear whether autogamy results from lack of pollinators, ineffective pollination or is caused by other factors.
Although biology and ecology of orchids, their pollination mechanisms, their nectar composition and its functions, along with information on the genus Epipactis are often described, the data are frequently inconsistent (Nilsson 1978;Brantjes 1981;Cingel 2001;Jakubska et al. 2005a, b;Jakubska-Busse andKadej 2006, 2008).
The study was designed to test whether autogamy in Helleborine is caused by lack of pollinators or whether it depends on specific insects species' that pollinate, how often the insects visit the flowers, and whether the flower's scent is a factor in the luring process.Additionally, nectar composition and the causes of geitonogamy were examined.South Moravia, the environs of Jihlava: Nove Syrovice, Kravsko, Vranovská Ves, Popůvky, Tetčice, Olbramkostel (see Fig. 1).

Insects behaviour
The insects were observed and photographed in natural conditions.In order to track insects' activity, the inside of the flower was marked with fluorescent pigments Radiant colour N.V. type TP33 (orange) and TP10 (chartreuse).
In order to determine the size of autogamy, cotton nets were put on selected inflorescences in specific populations well before the flowering period, which made the flowers inaccessible to pollinators.Next, the number of fruit produced by the plants was recorded in August.

Chemical analyses
Nectar was collected from flowers by using capillary glass with methylene chloride (GC Grade Merck) or n-pen-tane (GC Grade, Merck) as eluents, and stored in glass vials.
Then both extracts were condensed to 1ml volume using a rotary vacuum evaporator.The samples were analyzed using Perkin Elmer Gold TurboMass spectrometer with electron impact ionization EI and Perkin Elmer Autosysytem XL gas chromatograph equipped with an autosampler.The injector temperature was 320°C.
An Elite 5 MS column (5% phenylpolysiloxane) was used for the analyses (30 m long, inner diameter 0.25 mm, film thickness 0.25 µm, Perkin Elmer).Helium was used as the carrier gas at a flow rate of 0.8 ml/min, which was maintained by an electronic pneumatic control.
The GC oven temperature was held for 5 min at 40°C, then increased by 12°C/min to 300°C and held for 5 min.The temperature of MS transfer line was 250°C and the ion source worked at 180°C.The mass spectra were taken at electron energy 70 eV, with a scanning speed of 1 scans per 0.1 s from m/z 20 to 400.
The GC-MS data were processed using the TurboMass Software v. 5.4.2.Component identification was carried out using the NIST 2.1.0,NBS and WILEY mass spectral database.
Each analysis was performed in triplicate to assess the reproducibility of results.
The analysis of nectar chemical composition was done with consent from the Minister of the Environment, no.DONOOŚogiz-4211/I-6/129/08/ep.

Field study
The experiment was performed in field conditions.In order to test how insects react to flower morphology, colour pictures of the examined species were placed in close proximity to the studied ramets.This procedure allowed for determining the significance of flower physical qualities (such as its colour and shape) versus chemical attractants secreted by orchids in luring the insects.
Figure 2 shows a list of chromatograms TIC GC/MS.Surprisingly, E. atrorubens and E. palustris nectars have scents similar to each other but different from that of E. purpurata.It is possible that vanillin and its derivatives overwhelm other compounds constituting the scent (see Fig. 2).
The results only confirm that species of Epipactis in Central Europe are pollinated mainly by Vespidae and Apidae.
A strong tendency towards autogamy in small populations or populations which have a limited access to pollinators (e.g.dark forests) was found.In open populations such tendency was not found.The results partially confirm the Burns-Balogh et al. (1987) hypothesis that "autogamy is a pre-adaptation towards colonizing new habitats".
Another hypothesis stating that ants might be effective pollinators of Epipactis species has not been proven.Ants, mentioned by many authors (e.g.Peakall and Beattie 1989) as pollinators, rarely participate in pollination and should rather be treated as a potential vector.In Central Europe, the presence of ants, as well as their minute size, should rather be related to collecting the pollen as a food source.They are also drawn to orchids because of the nectar or honeydew produced by aphids living on orchids' shoots, both gathered for nourishment (see Fig. 3C and E).Ants, however, are main pollinators of tropical orchids such as Leporella fimbriata (Orchidaceae), the Australian selfcompatible orchid that is pollinated by winged male ants (Myrmecia urgens), which are sexually attracted to the flower and pseudocopulate with it (Rico-Gray and Oliveira 2007).Another Australian orchid, Microtis parviflora, is pollinated by flightless worker ants of the Iridomyrmex gracilis species complex (Peakall and Beattie 1989).
The study has shown that pollination biology varies greatly depending on the environmental factors as the air temperature and the amount of precipitation in the flowering season greatly influenced the results (Jakubska-Busse and Kadej 2008).Consecutive studies conducted on the same population over the course of several seasons revealed considerable differences in the quantity and the quality of pollinators.These differences are attributed to weather conditions.
The observations recorded during the study point to Diptera (Empis sp., Episyrphus sp.) and Hymenoptera (Apis mellifera) as the main pollinators of Epipactis palustris (see Fig. 4).It is likely that the numerously noted Calyptrata sp.along with other Diptera could also be pollinators.The presence of Meligethes aeneus (Coleoptera: Nitidulidae) should not, however, be directly related to pollinating Epipactis palustris due to a small body size of these beetles (Jakubska et al. 2005b).
The long list of insects (see Table 1) that have been found to frequently visits the flowers as well as the rich in attractants nectar composition (see Fig. 2) suggest that autogamy in this species is not caused by lack of potential pollinators or a poor luring strategy.
To test the range of autogamy, i.e. recurring visits to the same flower, and geitonogamy, (recurring visits to the same inflorescence), the flowers' inside of chosen specimens of E. atrorubens, E. palustris and E. purpurata was covered with fluorescent pigments to colour their visitors, which then allowed tracking their trajectory and behaviour.The insects prefer shoots with fresh flowers and visit them frequently which points to nectar consistency and intensity of secreted fragrance as strong attractants.The withering flowers or ones with dried nectar are far less attractive to insects and thus less often visited.The marked insects were also followed and observed on other plant species neighbouring the orchids, e.g.Campanula rapunculoides (Campanulaceae) and Chamaenerion angustifolium (Onagraceae).Some insects, including Bombus sp., walk along the inflorescence, starting on the bottom and moving upwards.They usually visit several flowers and repeat that pattern regardless of the plant species they visit.In conclusion, geitonogamy results from pollinators' biology (mainly Vespidae and Apidae), and is not related to Helleborine floral structure.
In open populations of all studied taxa, where other floral plants co-occur with orchids and serve as an alternative source of food for pollinators, autogamy was relatively rare.
Similar results were observed in the studies on pollination biology of E. atrorubens.The species was much often visited by insects during a warm and dry summer than during a humid one, which is due to the fact that insects do not fly in the rain, and that the emission of attractants is limited during rainfall and at low temperatures.
Since insects are poikilotherms and their activity depends on ambient temperature, Bombus sp.appears to be the main pollinator of Royal Helleborine (see Fig. 3).We found that bumblebees appeared on flowers the earliest with visits peaking between 10 a.m. and 2 p.m.
It confirms that they fly out to feed at approximately 10°C, and thus they are able to pollinate flowers during cold and rainy seasons.Bumblebees are more effective and more hardworking than honeybees.The high frequency of bumblebee's visits to orchids mentioned in other published research reports can be attributed by this bio-physiological adaptation.ACTA SOCIETATIS BOTANICORUM POLONIAE TABLE 1.The main pollinators of selected species of Epipactis Zinn, 1757 in comparison to Epipactis helleborine (L.) Crantz.

Species of Epipactis
List of pollinators and concomitant insects Xerothermic habitats of E. atrorubens are easily accessible to pollinators; additionally, the pollinators are lured by chemical attractants rich nectar.Only in populations close to the seaside, a lower frequency of insect visits was observed, which might be due to a deterring influence of strong and cool winds blowing from the sea.Additonally, the ramets often grow on exposed beaches, subject to intensive sun activity and thus their nectar evaporates faster than in specimens growing in shaded areas.As a result, the ramets become less attractive to insects relatively sooner.
The most interesting and, at the same time, the most controversial species among the examined ones is E. purpurata Sm., which grows with limited access to the light and without surrounding blooming plants, which could be a possible food source for potential pollinators or visitors, such as bumblebees (see Fig. 3F).In fact, bumblebees occur mainly in a close proximity to plants that serve them as a food source, e.g.Chamaenerion angustifolium (Onagraceae), Lythrum salicaria (Lythraceae), Vicia sp.(Fabaceae).In the habitats of E. purpurata these species were not present, which could explain low frequency of bumblebee's visit.
The differences in nectar chemical composition and the flowers fragrance in E. purpurata (see Fig. 1) are hard to determine.The procedure involving the nets, which were supposed to help in assessing the level of autogamy, indicated that the species produced over 82% of flowers and proved that the plant is autogamic.Although the orchid successfully produces nectar rich in attractants, the potential pollinators rarely occur in its habitat which in the end results in less insects visiting the flowers.Therefore, it is possible that autogamy of this species is connected to its limited access to pollinators.Among the examined populations E. purpurata produced only flowering shoots, and no vegetative ramets.The only known pollinators of this species are: a male cuckoo wasp (Hymenoptera, Chrysididae), and a hoverfly, Episyrphus balteatus, which has also been recorded visiting it (Anonimus 2010).Another poten-tial pollinator of E. purpurata is Coccinella, as it was observed to accidentally carry pollen.

Floral morphology vs. chemical attractants
The research procedure involving displaying pictures of orchids, was designed to determine whether different shapes and colours of flowers influence the frequency of insects visits.No such relationship, however, was discovered.According to other sources, red flowers should be attractive to flies, but these were equally often seen on white flowers of E. palustris or blue ones of E. purpurata.Therefore, it seems that the key role in luring biology belongs to chemical attractants.
It was observed that the insects usually did not return to the marked flowers, which can be explained by the presence of other food sources (i.e.melliferous plants) in close proximity to the examined orchids.
The influence of nectar components on insects' behavior posess another fascinating question.The obtained results partially confirm suggestions of Brodmann et al. (2008) on the significance of such compounds as nonanal, decenal, benzaldehyde in the process of luring the insects, particularly Vespidae.Yet, it seems that there are more compounds in the nectar that could influence insects' behaviour, as not only representatives of Vespa or Apis genera but also other creatures were observed to visit the plants.Furthermore, it should be remembered that nectar is a complex composition of compounds and their derivatives.
The composition of the scent released by Epipactis palustris might be the most crucial in initially attracting pollinators and visitor insects.The compounds that were discovered, such as eugenol and vanillin, are strong aromatic attractants as a substantial number of Diptera representatives, which have a sense of smell, visited flowers of E. palustris.Among Diptera, Episyrphus balteatus (Diptera: Syrphidae) was found to be a pollinator of Epipactis thunbergii (Cingel 2001).Furthermore, it seems that Episyrphus is a universal pollinator of all flower plants.Anomala dubia (Scarabaeidae) and Gaurotes virginea (Cerambycidae) and Diptera -Calyptrata sp.(Muscidae) beetles might be pollinators of E. palustris, as they have a desired body size and were often seen on the flowers in question.Yet, no pollen was seen to stick to the bodies of aforementioned beetles.Orchids visited and pollinated by insects create perfect living and hunting conditions for spiders and predatory froghoppers, which feed on pollinators.The following Heteroptera: Liocoris tripustulatus (Miridae), Lygus pratensis (Miridae) and Arachnidae species Metellina mengli (Tetragnathidae) and Philodromus dispar (Philodromidae) were repeatedly observed on orchids.Interestingly, Misumena vatia (Araneae) very often landed on the displayed flower pictures and always headed toward its bottom, as it would do on real flowers.
The procedure that introduced isolating inflorescences in order to prevent the flowers from being visited by insects brought an even more surprising conclusion -namely, 74.9% of all examined Helleborine flowers produced fruit, which proves the domination of autogamy.The percentage of autogamy was the highest in E. purpurata 82% and in E. palustris 79.4%, as opposed to the lowest in E. atrorubens 63.5%.However, the findings could be distorted by the qualities of the studied populations.